Hemoglobin is a naturally occurring protein. When contained in red blood cells, hemoglobin exists in the form of a tetramer of four sub-units, two .alpha. sub-units and two .beta. sub-units. Each sub-unit has a protein chain and a heme molecule, the protein chains of the .alpha. sub-units containing 141 amino acids of known sequence and the protein chains of the .beta. sub-units containing 146 amino acids of known sequence. As a natural, bio-acceptable and well studied protein, hemoglobin is an attractive candidate for transport of bio active materials, drugs and other species in vivo, by attachment of such species to hemoglobin and introduction thereof into the circulatory system. Hemoglobin in solution outside the natural red blood cell, however, tends to dissociate from the tetrameric form into dimers and even monomers of the sub-units. These have too low a molecular weight to be adequately retained in circulation in the body. Accordingly, formation of chemical crosslinks between the sub-units of hemoglobin, to stabilize it in its natural tetrameric form, and hence ensure its adequate retention in the circulatory system, is necessary before hemoglobin can be developed as such a transport system.
In order to render a hemoglobin product properly acceptable in biomedical applications, control over its precise chemical nature is needed. Accordingly, it is desirable that any crosslinking which is effected between sub-units of hemoglobin is by means of specific and reproducible chemical linkages between specific sites or chemical groups on the hemoglobin molecules. With protein chains the size of those of the sub-units of hemoglobin, each containing substantial numbers of reactive primary amino groups, for example, this is difficult to achieve.